Expression systems for the production of biological therapeutics or biopharmaceuticals, such as recombinant proteins, generally consist of a nucleic acid vector construct encoding the desired recombinant therapeutic and a chosen host cell. The vector is introduced into the host cell and the endogenous cell machinery is utilised for the production of the desired therapeutic i.e. the desired recombinant protein. The intricacies in establishing an efficient and reliable expression system for the production of approvable biological therapeutics are manifold. However, well-established expression systems may provide cost-effective alternatives for the production of pharmaceutical products otherwise difficult to obtain.
Efficiency of the system itself depends on a large variety of factors including the design of the vector and the choice of host cell. The strategic combination of regulatory elements, selection markers and stability elements within the vector sequence have to balance simple manipulation and application of the vector with high yield production of the desired biological therapeutic. Determining a cell's suitability to act as host cell in such an expression system is primarily governed by the need to maximise compatibility between the endogenous cell machinery and the regulatory elements present in the vector, while keeping potential adventitious contaminants in the final product minimal. Further, availability, cost and acceptability for regulatory approval of any therapeutic produced by the system, have to be considered.
Nucleic acid vectors used in expression systems comprise plasmids, cosmids, Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), retroviral. adenoviral and lentiviral vectors. These vectors differ in many characteristics, such as their capacity to accommodate different sized nucleic acid inserts. their most efficient introduction method into the host cell and specifically in their utilisation of the endogenous cell machineries of different types of host cells to ensure sufficient expression of the desired protein.
Regulatory elements commonly present in such expression vectors influence transcription. translation as well as protein synthesis of selection markers and of sequences encoding the desired biological therapeutic. Such regulatory elements include, but are not limited to, promoters, terminators, modifiers, insulators, spacers, regulatory protein binding sites, introns, inducers, etc.
Known promoters include constitutively active promoters such as the thymidine kinase (TK) promoter, the actin promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, the simian vacuolating virus 40 (SV40) early promoter, the cyclin T1 promoter, the RNA polymerase III U3 promoter, the cyclophillin promoter, the cytomegalovirus (CMV) promoter, the Autographa californica nuclear polyhedrosis virus (AcNPV) P10 promoter and the β3-galactosyltransferase 5 (β3GAL-T5) promoter.
Known promoters also include inducible promoters such as the heat shock protein 70 (HSP70) promoter (stress induced), the heat shock protein 90 (HSP90) promoter (stress induced), the alcoholdehydrogenase I (alcA) promoter (alcohol induced), the activating copper-metallothionein expression (ACE1) promoter (metal induced), the small subunit of ribulose-1,5-bisphophate-carboxylase (SSU1) promoter (light induced), the hypoxia induced factor 1α (hif1α) promoter (hypoxia induced), the inducer of meiosis 2 (IME2) promoter (starvation induced), the glucocorticoid receptor (hormone induced), the estrogen receptor (hormone induced) and the ecdysone receptor (hormone induced).
Further, cell type/tissue specific promoters, such as the nkx2.5 promoter (heart cells), the islet 1 promoter (pancreatic cells), the MyoD promoter (muscle cells), the cluster of differentiation 2 (CD2) promoter (T-cells) and the collagen II promoter (cartilage), are known to change their level of activity in response to cell type specific stimuli or to progression through developmental stages.
Known terminator elements, such as the RNA Polymerase II terminator, the small nucleolar RNA 13 (snR13) terminator, the bovine growth hormone (BGH) terminator, the simian virus 40 (SV 40) terminator and the thymidine kinase (TK) terminator, may provide suitable polyadenylation signals.
Known modifier and insulator elements include the tetracycline operator/receptor (tetO/tetR) system, the upstream activating sequence of the galactose dependent GAL4 transcription factor (GAL4 UAS), the adenovirus early region B1 TATA box, binding sites for the herpes simplex virus (HSV) regulatory protein VP16, the 5′HS4 chicken β-globin insulator, the paternally expressed gene 3 (Peg3) insulator and the sea urchin arylsulfatase (ARS) gene insulator.
While many attempts have been made to establish efficient and reliable expression systems for the production of approvable biological therapeutics, problems relating to low yield and adventitious contamination of the produced biopharmaceuticals remain. Choosing the most effective combination of suitable regulatory elements from the plethora of options, such that the system conveys stability and the highest degree of compatibility with the endogenous host cell machinery, poses a major challenge in the field.
Obtaining regulatory approval for a biopharmaceutical product poses a further challenge. Regulatory approval involves determination of the safety and efficacy of the pharmaceutical product prior to marketing. The process of gaining regulatory approval for innovator drugs is very time consuming and expensive. However, once approved, these drugs may be very profitable. particularly when they are marketed under exclusivity rights such as patent protection.
The profitability of innovator drug's market may provide a substantial incentive to exploit this market once patent rights have expired. Following patent expiry, innovator drugs can be marketed as generic drugs or biosimilars for drugs produced by recombinant DNA technology. Generic versions of blockbuster biopharmaceuticals near patent expiry include Epogen (erythropoietin, EPO) and Neupogen (granulocyte colony stimulating factor, G-CSF). The approval of a follow-on version of Pfizer's Genotropin (recombinant human growth hormone) seems to indicate a change in a landscape where previously, biopharmaceuticals enjoyed immunity from competition even after expiration of their patent protection. At present, there are over 80 generic versions of biopharmaceuticals in development (Datamonitor 2010).
Biosimilars ideally are bioequivalents of the innovator drugs and, as such, the path to regulatory approval for biosimilars is in theory less arduous than for the original innovator drug as the clinical data establishing safety and efficacy have been carried out.
Approval of generic biopharmaceuticals is dependent on comparable dosage form, strength, route of administration, quality. performance characteristics and intended use compared with approved biopharmaceuticals (that is, the reference listed drugs). For example, under the United States Food and Drug Administration (FDA), approval for a generic drug involves an “Abbreviated New Drug Application” (ANDA) which generally does not include pre-clinical and clinical data to establish safety and effectiveness. Approval also involves a bioequivalence review, which establishes that the proposed generic drug is bioequivalent to the reference-listed drug. This bioequivalency is based upon a demonstration that the rate and extent of absorption of the active ingredient in the generic drug fall within the scope of the parameter of the reference listed drug.
Importantly, there is a chemistry/microbiology review process that provides an assurance that the generic drug will be manufactured in a reproducible manner under controlled conditions to ensure that the drug will perform in a safe and acceptable manner.
Although guidelines for the approval of biosimilar drugs exist, there is uncertainty in regard to the practicalities of regulatory approval of biosimilars. Much of the uncertainty is driven by the lack of a clear practical and detailed regulatory pathway for the approval of such drugs and the scientific debate over product comparability and interchangeability. The uncertainties resulting from the manufacture of biosimilar drugs under conditions different than those used by the innovator suggest that it may be impossible to develop a true “generic” version of a biotechnology drug. Indeed, regulatory authorities in Europe and the US have shunned the use of the term “biogeneric”, preferring the nomenclature “biosimilar” and “follow-on biologicals”.
Many quality concerns for expression system-derived biopharmaceuticals have originated from the presence of adventitious contaminants or from the properties of the host cells used to prepare the product. Several of these products have also had quality concerns regarding the expression vector of the system. It is well established that cell properties and events linked to cell culture can affect resultant product quality and safety. Effective quality control of recombinant products requires appropriate controls on all aspects of handling the cell and cell culture. This is particularly relevant to the development of biosimilars.
Previously, Chinese Hamster Ovary (CHO) cells were modified and engineered to produce insulin. However these CHO cells were unable to express biological active insulin and thus the patents associated with this type or method of CHO cell modification were not commercial exploited. Fully functional insulin was not produced in CHO cells due to cryptic splicing of the insulin gene message by the translational machinery In the CHO cell.
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.